This invention relates interactions between genotype and diet in swine that prevent E. coli associated intestinal disease. Methods and compositions are provided for identifying swine which are genetically able to utilize low cost feed stuffs and feed compositions to prevent intestinal disorders after weaning.
Disease caused by F18+E. coli can be prevented by reducing the amount of soy bean meal (SBM) and total protein in the diet; however, this decrease in the total protein content of the pig""s diet results in decreased quality e.g. weight (Bosworth et al., 1996; Bertschinger et al., 1979). Therefore, this treatment for F18+E. coli is not economical. Diets that prevent disease due to F18+E. coli but still contain high enough protein levels to allow swine to gain weight rapidly after weaning would be desirable.
Reduction of costs in swine production is a goal of breeders. Two of the most significant costs associated with swine production relate to food and disease, especially in the postweaning period. Intestinal diseases reduce weight gain, hence decrease the value of the swine. Treatment costs also occur. Swine weaned early require a high amount of protein in their diet in order to gain weight adequately, and the source of the protein is a major food diet cost. Plant-based sources high in protein content, typically soy bean meal (SBM), are common ingredients in most swine diets and are cheaper than animal-based sources, such as fish meal and blood meal. However, swine fed diets with high levels of SBM frequently have a reduced rate of weight gain relative to swine fed nursery diets postweaning in which a portion of the SBM is replaced with animal byproducts, such as whey and plasma (Li et al., 1991). The mechanisms responsible for this reduced rate of gain in recently weaned swine are not defined, but it could be that SBM acts as a food allergen in recently weaned swine who have just been removed from a diet high in animal protein (i.e., sows"" milk).
A major problem in breeding swine is to keep them disease-free. Intestinal disorders postweaning are a particular problem. Intestinal disease is manifest in swine infected with F18 E. coli who may get either diarrhea and/or edema disease after weaning. Newborn swine are resistant to disease due to F18 E. coli because they do not express intestinal receptors for this E. coli and thus, the E. coli is unable to adhere to and grow (colonize) the intestine of newborn swine (Nagy et al, 1992). F18 E. coli can also cause a mild subclinical disease, which results in a decreased rate of gain after weaning, but may cause no other detectable clinical signs (Bosworth et al., 1996). Swine suffering from subclinical disease also have very mild vascular damage in the brain and intestines, problems only detectable by thorough histologic examination performed by an experienced investigator.
A limited number of serotypes of toxigenic Escherichia (E.) coli strains are the causative agents of oedema disease and postweaning diarrhea in swine which induce serious economic losses, especially among piglets aged 4 to 12 weeks, in swine breeding farms all over the world. The typical clinical symptoms of oedema disease are neurological signs such as ataxia, convulsions and/or diarrhea. At post mortem examination, oedema is typically present at characteristic sites such as eyelids and forehead, stomach wall and mesocolon. The diseases are caused by Shiga-like toxin-II variant and enterotoxins LT, STa, STb respectively, produced by E. coli that colonize the surface of the small intestine without effecting major morphological changes of the enterocytes (cells in the intestine). Types of bacterial E. coli strains, such as F4, F18 and K88 are major lethal villains in this regard. xe2x80x9cOedema disease of swine is an enterotoxaemia characterized by generalized vascular damage. The latter is caused by a toxin, Shiga-like toxin II variant, produced by certain strains of E. colixe2x80x9d (Bertschinger et al., 1993). The E. coli are distinguished by their pili types, a group of adhesive fimbriae that are related are designated e.g., K88 or F18 (Vxc3x6geli et al., 1996).
Not all swine succumb to E. coli infections. Colonization depends on adherence of the bacteria to the enterocytes which is mediated by the bacterial fimbriae designated e.g., K88 or F18. Susceptibility to adhesion, i.e. expression of receptors in swine for binding the fimbriae, has been shown to be genetically controlled by the host and is inherited as a dominant trait with, in the case of F18, B being the susceptibility allele and b the resistance allele. (Vxc3x6geli et al., 1996; Meijerink et al., 1996). The genetic locus for this E. coli F18-receptor (ECF18R) has been mapped to porcine chromosome 6 (SSC6), based on its close genetic linkage to the S locus and other loci of the halothane (HAL) linkage group on chromosome 6. The receptor for K88 E. coli is on chromosome 13.
The mechanism for resistance appears to be that intestinal borders in resistant animals are not colonized by E. coli, i.e., the bacteria do not adhere to intestinal walls of resistant swine. Glycoprotein receptors in the brush border membrane of the intestine were shown to be responsible for the differences between adhesive and non-adhesive phenotypes related to some E. coli, therefore, the genotype of the host swine determines resistance. The fimbriated bacteria also have been studied. (WO 9413811)
Current methods of identifying swine that are resistant to F18 E. coli associated diseases are either to 1) collect intestinal samples from swine at slaughter and perform the microscopic adhesion test, (see Example 2 herein) 2) challenge the animals with virulent E. coli (xe2x80x9ccolonization testxe2x80x9d), or 3) perform blood typing of the A-O(S) blood group system. The first two methods are not practical for identifying resistant animals for use as breeding stock. Although the blood typing method does identify resistant animals, the test is unable to determine whether susceptible animals are homozygous or heterozygous for susceptibility. At least two alleles (condition of a gene) at the receptor locus control either susceptibility (dominant) or resistance (recessive). Knowledge of the genotype of animals with regard to these alleles is essential to develop a successful breeding program. The purpose of the breeding program is to produce swine that are resistant to F18 E. coli associated diseases that decimate stock post-weaning.
In one publication the authors stated, in reference to oedema disease in swine, that xe2x80x9cSearches are underway for appropriate genetic markers . . . xe2x80x9d (Bertschinger et al., 1993, page 87) and, citing Walters and Sellwood, 1982:
Breeding resistant swine is an attractive method for prevention of diseases for which an effective prophylaxis is not available. The feasibility of this approach will depend on the prevalence of the gene(s) encoding resistance in the pig population, improved methods for the detection of resistant swine, and absence of negative genetic traits co-selected with this resistance.
A genetic xe2x80x9cmarkerxe2x80x9d locus is a coding or non-coding locus that is close to a genetic locus of interest, but is not necessarily the locus itself. Detectable phenotypes include restriction length fragment polymorphisms, colorimetric or enzymatic reactions, and antibiotic resistance. The S locus controls expression of the A and O blood group antigens. Swine homozygous recessive at the S locus do not express either A or O blood group antigens. A similar condition exists in humans and is due to mutations in the alpha (1,2) fucosyltransferase gene which encodes the human blood group H (Kelly et al., 1994; see also WO 9628967). The porcine alpha (1,2) fucosyltransferase gene of swine has recently been sequenced (Cohney et al., 1996). This gene is very likely the gene present at the S locus in swine.
The blood group H and Se loci have been mapped genetically and physically to human chromosome 19q13.3. This region is evolutionarily conserved, containing genes homologous to the HAL linkage group of genes in swine. The blood group H encoding gene is the so called FUT1 whereas the Se gene is equivalent to the FUT2 gene. FUT1 determines H antigen expression in the erythroid cell lineage, whereas FUT2 regulates expression of the H antigen in the secretory epithelia and saliva. Conservation of the FUT1 gene has been shown in lower mammals such as rat and rabbit, and mRNA expression has been shown in rabbit brain tissue and rat colon. In all these species two types of alpha (1,2) fucosyltransferase genes have been reported which are structurally very similar to the human FUT1 and FUT2 genes, but in particular the FUT1 homologous genes show a species specific expression pattern. In humans the FUT1 gene is responsible for synthesis of H antigens in the precursors of erythrocytes. However, in swine erythrocytes passively adsorb H-like antigens from the serum, as is the case for the human Lewis antigens. In swine all H-like antigens are related to exocrine secretory tissues, and expression of the FUT2 (Secretor) gene is seen in secretory tissue of other animal species. Therefore, expression of the porcine A-O blood group determinants which cross-react with anti-human blood group H and A antibodies might be influenced by the FUT2 gene.
Further information about blood groups and E. coli swine diseases include that carbohydrate structures of blood group antigens mediate the adhesion of some pathogenic microorganisms to host tissues, e.g. Helicobacter pylori adhere to Lewisb blood group antigens, and E. coli causing urinary tract infections adhere to blood group P substance. Genes encoding glycosyltransferases that are responsible for the formation of the blood group specific carbohydrate structures, therefore, represent candidate genes for the control of bacterial colonization by the host. The localization of these genes is in the same chromosomal region as the locus responsible for adhesion/non-adhesion of F18 positive E. coli in the swine small intestine. Swine do not express blood group antigens A and O until after weaning, this is the same time that they become susceptible to disease caused by F18 E. coli. 
New methods of diagnosis and treatment are needed for E. coli related intestinal diseases in swine. Detection of a genetic mutation was proposed as a diagnostic test for some swine disorders (malignant hyperthermia) (Fujii et al., 1991; U.S. Pat. No. 5,358,649), but polymorphic markers were not reported for diagnosis. Vaccines to develop resistance to E. coli colonization were described (U.S. Pat. No. 5,552,144; WO 8604604), but are unlikely to be a preferred method to prevent the E. coli disease because of difficulties in administering live vaccine orally to newborn swine, and because of regulatory restrictions. Antibiotics are available for treatment, but there is no successful prophylaxis.
Compositions and non-invasive methods are available for the identification of swine genetically resistant to E. coli related diseases, in particular, intestinal diseases associated with a strain of E. coli bacteria supplied with F18 fimbriae. DNA polymorphisms in the swine alpha (1,2) fucosyltransferase (FUT1) gene were identified that differentiate resistant from susceptible swine and provide a diagnostic test useful for swine breeders. However, further improvements in disease control and weight gain are desirable.
Understanding and controlling the decreased rate of gain in swine fed diets with high levels of SBM would be economically beneficial to the swine industry. If subclinical disease due to E. coli infection is the reason for the decreased rate of gain in swine fed high levels of SBM, preventing or controlling this subclinical disease would be useful.
It has not been determined if diets in which SBM has been replaced with animal-based proteins can prevent subclinical and/or clinical disease due to F18+E. coli or if the decreased rate of gain seen in swine fed diets with high levels of SBM is due to subclinical disease. Additionally, it is not known if diet and genetics can interact in preventing disease due to F18 E. coli. 
A method for improving weight gain in swine has the following steps: selecting swine that are genetically resistant to E. coli colonization and feeding the selected swine high levels of a plant-based protein concentrate, wherein high levels are generally about 20% or greater. A suitable plant-based protein concentrate is SBM. The selecting includes determining in a biological sample from the swine whether or not adenine is the only nitrogen base at position 307 in alpha 1,2 fucosyltransferase gene, and feeding the swine of this genotype high SBM diets. A further method of preventing F18 E. coli colonization in swine is by replacing some or all of the plant-based proteins in the diet fed to the swine, with animal-based proteins. Suitable animal-based proteins include milk, plasma and fish meal. This is particularly efficient for swine genetically susceptible to F18 E. coli colonization, that is, swine that do not have only adenine as the nitrogen base at position 307 in the swine alpha 1,2 fucosyltransferase.